Studies on plasmid replication have contributed significantly to our understanding of the regulation of DNA replication. Since bacterial plasmids are small DNA molecules that replicate autonomously, they serve as useful systems for studies on the mechanism as well as regulation of DNA replication. Plasmids are intimately associated with antibiotic resistance and toxin production in a number of pathogens. Hence studies on plasmids are of considerable medical significance as well. We are investigating the mechanism and regulation of replication of a small Staphylococcus aureus plasmid, pT181. This plasmid is 4.4 kb in size and codes for tetracycline resistance. We have isolated the pT181-encoded repC protein that is required for replication. The role of repC in initiation, elongation and termination will be investigated by using anti-repC antibodies to inhibit in vitro replication of pT181. By site-specific mutagenesis of the origin, nucleotides that are critical in replication will be identified. Mutants of repC will be isolated by site-directed mutagenesis and studied for their replication, relaxation and DNA-binding activities by filter binding, DNaseI foot-printing and in vitro replication experiments. Amino acids in repC that are critically associated with these activities will be identified. The NH2- and COOH-terminal domains of repC will be isolated by molecular cloning using expression vectors and their DNA binding properties determined by filter binding and DNaseI foot-printing. The mechanism of initiation of pT181 replication will be investigated by using the in vitro system. The topoisomerase-like activity of repC will be studied by determining the changes in linking numbers by repC during relaxation by agarose gel electrophoresis. Any possible nicking or double-stranded cleavage activity of repC will be identified by gel electrophoresis, density gradient centrifugation and DNA sequencing. If repC nicks at the origin sequence, this nick may correspond to the start-site of replication. If repC creates a site-specific nick in the origin, in vitro replication experiments will be carried out in the presence of dideoxy NTPs and the approximate start-site located by restriction analysis and gel electrophoresis or radioactively labeled replication products. A radioimmunoassay using anti-repC antibodies will be carried out to determine the amount of repC in a number of pT181 copy mutants and any direct relationship between amount of repC and copy number will be identified. Preliminary experiments will be carried out to determine if repC works as part of an initiation complex with other proteins.